The long-term objective of this research program is to understand how the sequence of a protein determines its structure, stability, folding kinetics, and ability to be denatured spontaneously and by enzymes. This is one of the central, unsolved problems in molecular biology and is important because protein folding is a prerequisite of most biological processes, and protein unfolding and misfolding often lead to disease. In addition, much of the promise of biotechnology for improving human health depends on our eventual ability to manipulate the function of proteins by altering their sequences and structures. We will probe fundamental issues in protein folding and design using P22 Arc repressor and "switch" Arc, a variant in which the b-sheet of the wild-type protein is replaced by a pair of helices. Arc provides an excellent model system for studying the folding and association of a single-domain dimeric protein using genetic, biochemical, and biophysical methods. Switch Arc provides an opportunity to study the sequence determinants of structural specificity and the stepwise evolution of a protein fold. These studies will be important for testing and refining our understanding of the sequence determinants of protein structure and stability, and for evaluating how well computational algorithms serve to explain and/or predict the effects of sequence changes on structure and stability. Some proteins and assemblies are too stable for spontaneous unfolding/dissociation to be a biologically relevant reaction, and energy-dependent disassembly chaperones play critical intracellular roles in dismantling such complexes, in solubilizing aggregates, and in mediating protein degradation. To begin to understand these processes, we will dissect the biochemical and enzymatic properties of C1pX-a hexameric-ring ATPase-which catalyzes protein denaturation and regulates the activity of the C1pXP protease. By studying C1pX transactions with a large number of different protein substrates, we will determine how the local stability and dynamical properties of these substrates affect enzyme-mediated denaturation. Enzymes related to ClpX are important virulence determinants in some pathogenic bacteria, play roles in regulation of bio-film formation, and function in key processes in mammalian cells, including proteasome-mediated protein degradation and vesicle fusion.